Disruption of SOCS1 Promotes the Antitumor Activity of CD19-Specific CAR NK Cells Coexpressing IL-15

Abstract

Background Chimeric antigen receptor (CAR) expressing NK cells exhibit robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. Interleukin-15 (IL-15) promotes NK cells expansion and survival, while IL-15 signaling is often inhibited by the suppressor of cytokine signaling (SOCS) proteins (CIS, SOCS1-7), which is rapidly induced in response to IL-15 and negatively regulates cytokine signaling through the JAK/STAT pathway in NK cells. We hypothesized that SOCS1 would be a logical checkpoint to target in human NK cells to increase their persistence and thereby enhance their antitumor potency. Here, we devised a strategy that couples targeting the SOCS1 protein, with CD19-specific human NK cells that constitutively express IL-15. Method Firstly, we evaluated the SOCS1 mRNA expression levels on NK cells after cultured with or without IL-15 for 6 or 24 hours by reverse transcription polymerase chain reaction (RT-PCR). The retroviral vector encoding CAR19.4-1BB-zeta-2A-IL-15 (named BBz.IL-15) and the production of transient retroviral supernatants have been previously described. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. CAR expression on NK cells was determined by flow cytometry, and IL-15 secretion was measured by ELISA. Knockout (KO) of SOCS1 by CRISPR/Cas9 using the Neon transfection system was performed on day 3 post transduction in both non-transduced NK cells (NT-NK) and CAR-transduced NK cells (CAR-NK). To assess KO efficiency, we used polymerase chain reaction (PCR) gel electrophoresis, western blot, and Sanger sequencing. The degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 7 post electroporation. Results The expression of SOCS1 on NK cells was significantly induced within 6 hours of IL-15 treatment. Our approach for combined retroviral transduction with the anti-CD19 CAR and RNP-mediated gene editing of SOCS1 showed that surface expression of the CAR was maintained on NK cells in culture, with construct containing BBz.IL15 (37.18 ± 11.71%) demonstrating comparable transduction efficiency compared with the BBz construct (45.48 ± 10.54%) (P＞0.05), and the efficiency of SOCS1 KO was high in both the nontransduced (NT) control and CAR-expressing NK cells by TIDE and remained stable over time. When tested against CD19-postive Raji cells, SOCS1 KO BBz.IL-15 NK cells produced more IFN-γ and TFN-α, and displayed greater degranulation (CD107a) against their targets than did their BBz.IL-15 NK controls (BBz.IL-15 SOCS1 KO vs BBz.IL-15 control: 15.75±2.05% vs 7.93±0.47% for IFN-γ, P=0.032; 6.27±1.27% vs 1.88±0.26% for TFN-α, P=0.04; 23.25±2.33% vs 12.35±2.33% for CD107a, P=0.042). However, there was no significant difference between the SOCS1 KO BBz. or NT NK cells and their respective BBz. and NT controls regarding IFN-γ and TFN-α production and degranulation (BBz.SOCS1 KO vs BBz.control: 16.95±5.30% vs 16.85±8.70% for IFN-γ, P＞0.05; 6.83±2.98% vs 4.06±2.47% for TFN-α, P＞0.05; 26.65±7.14% vs 25.8±2.12% for CD107a, P＞0.05) (NT SOCS1 KO vs NT control: 21.75±6.58% vs 7.5±2.18% for IFN-γ, P＞0.05; 7.23±3.46% vs 1.58±0.03% for TFN-α, P＞0.05; 15.05±3.46% vs 6.84±2.15% for CD107a, P＞0.05). To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. SOCS1 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS1 KO NK cells can grow faster than the control group. Conclusion In conclusion, the results demonstrate that SOCS1 might be a potential target for genetic modification to improve activity of CAR- NK cells.